Abstract

1.1 Background: Mitochondria are the main site of energy production in most cells. Furthermore, they are involved in a multitude of other essential cellular processes, such as regulating the cellular calcium pool, lipid metabolism and programmed cell death. Healthy and functional mitochondria are critical to meet the fundamental needs for almost all cell types, which makes mitochondrial quality control (QC) very important. Given the high energy demand of neuronal cells, their vulnerability to endo- and exogenous stressors, and their post-mitotic status, mitochondrial QC plays an important role in neuronal survival with failing mitochondrial quality control linked to many neurodegenerative diseases such as Alzheimer's disease and many others. Several well-orchestrated mechanisms of mitochondrial QC are in place to maintain mitochondrial function and to prevent cellular damage through dysfunction of mitochondria. Depending on the severity of mitochondrial damages, mitochondrial QC takes place on the molecular level, organelle level, and/or cellular level. Through those mechanisms damaged proteins are degraded, damaged mtDNA is repaired, and depolarized mitochondria are recycled. However when the first two mechanisms fail to reverse the damage, cellular level QC in form of programmed death or apoptosis takes place to limit further damage resulting from dysfunctional mitochondria. Mitochondria are highly dynamic organelles that consistently undergo fusion and fission processes. Mitochondrial dynamics is a central part of mitochondrial quality control as mitophagic recycling of damaged mitochondria and as execution of cell death relies on the functional mitochondrial fission machinery. Ubiquitin-dependent proteasome-mediated protein degradation was recently implicated in mitochondrial QC as well as in the regulation of mitochondrial morphology. As part of the ubiquitin-proteasome system, (Membrane-Associated-Ring-CH 5) RING finger (Really-Interesting-New-Gene)-domain containing transmembrane ubiquitin E3 ligase MARCH5 was shown to localize to the outer mitochondrial membrane (OMM). In addition, a role for MARCH5 in regulating mitochondrial dynamics was demonstrated through the ubiquitin-modification of dynamin-related-protein 1 (Drp1) and some others. Interestingly, inactivation of MARCH5 resulted in mitochondrial elongation consistent with a role of MARCH5 in the regulation of Drp1-mediated mitochondrial fission. This feature correlates mitochondrial QC with the ubiquitin-proteasome-system (UPS). Furthermore, MARCH5 was shown to be involved in the degradation of the amyotrophic lateral sclerosis-related protein mSOD1 and the Joseph-Machado-disease protein ataxin 3. Thus, MARCH5 function is likely involved in maintaining mitochondrial health in neuronal cells. In order for proteasomal degradation of mitochondrial proteins to take place, protein retrotranslocation from the mitochondrion to the cytosol where the proteasome resides is needed. Recently, the AAA-ATPase p97/VCP/Cdc48, an essential part of the ubiquitin-proteasome system known to facilitate the retrotranslocation of ubiquitylated proteins for further degradation, was shown to be involved in mitochondrial QC. While the role of p97 in mitochondrial protein QC is far from being understood, mutations in p97 were reported to be involved in Inclusion Body Myopathy with Paget Disease of Bone and/or fronto-temporal Dementia (IBMPFD). 1.2 Aims of the study: The main aim of this study was to further explore and elucidate mechanisms of ubiquitin proteasome system-mediated mitochondrial proteostasis and quality control. Despite a lot of progress in the last several years, the involvement of outer mitochondrial membrane- localized ubiquitin ligases such as MARCH5, and their involvement in OMM proteins degradation is still not well understood. Thus, a particular aim of the study was to further characterize the involvement of MARCH5 in mitochondrial maintenance. Next to ubiquitin ligases, protein retrotranslocation was proposed to be important for mitochondrial maintenance with the AAA-ATPase p97 recently connected to this process. Thus, the characterization of potential mitochondrial roles of p97 has been the focus of the next goal, in order to investigate the potential capacities of this multifunctional AAA-ATPase in mitochondrial membrane associated protein degradation. A last aim was to further connect mitochondrial quality control to mitochondrial function in neuronal-like cells and to better understand the role of ubiquitylation-dependent protein degradation in maintaining the health of neuronal-type cells especially under low stress conditions. To this end we investigated mitochondrial behavior and functional alternations in neuron-like cell lines in response to inactivation of MARCH5- and p97-dependent mitochondrial QC mechanisms. 1.3 Methods: As model for neuronal cells, two different neuron-like cell lines were used during the experiments, namely RGC5 (mouse neuron-like cell) and SH-SY5Y (human neuroblastoma) cells. In order to analyze the ubiquitin-mediated, proteasome-dependent degradation of mitochondrial proteins under normal and low stress conditions, the activity of two proteins, MARCH5 and p97, shown to be involved in mitochondrial protein degradation was blocked. To this end, cell lines expressing dominant-negative mutants of MARCH5 (MARCH5H43W ) or p97 (p97QQ ) were generated by transfection and selection for stable expression. In order to model low level neurodegenerative stress to neuronal cells below the mitophagic and apoptotic threshold but likely to induce mechanisms of mitochondrial proteostasis, neuron-like cells were exposed to conditions of elevated pressure or hypoxia/re-oxygenation or were treated with the known neurotoxic agents 6-hydroxydopamine, the mitochondrial complex I inhibitor rotenone, or the Alzheimer’s related Aß peptide. Confocal microscopy was used to acquire high solution images of mitochondrial morphology, while quantification of mitochondrial fusion and fission dynamics was performed by using a mitochondrial matrix-targeted photoactivatable-GFP (mtPA-GFP)-based fusion assay. In addition, confocal life cell imaging and flow cytometry was employed to analyze other mitochondrial functional parameters, such as mitochondrial membrane potential, mitochondrial reactive oxygen species (ROS) production, and cellular survival. A modified western blot technique (OyxBlot) was applied to demonstrate oxidized protein levels on isolated mitochondria. Unpaired, two-tailed Student’s t-test, and paired t-test with Holm adjustment for multiple comparisons were used to analyze the data according to different specific data sets. For a detailed description of the employed methods please refer to the material and methods section of the included manuscripts. 1.4 Results: Neuron-like RGC5 cells exposed to neurodegenerative stress in form of increased hydrostatic pressure, hypoxia-reoxygenation, and electron transport chain inhibition following rotenone treatment showed increased mitochondrial fragmentation rate, increased cytochrome c release and cell death. Expression of MARCH5H43W alleviated the mitochondrial damage following exposure to stressors, by significantly (p<0.05) reducing mitochondrial fragmentation, inhibiting mitochondrial cytochrome c release, and preventing RGC5 cell death, in comparison to MARCH5 expressing cells. These effects were shown to be similar to Drp1 inactivation, which was previously shown to be involved in mitochondrial fission and the execution of cell death. Furthermore, neuron-like SH-SY5Y cells exposed to neurotoxic stressors such as rotenone, 6-hydroxydopamine, and amyloid-beta peptide were employed to investigate the role of MARCH5-mediated QC in neuronal cells. As for SH-SY5Y cells, expression of MARCH5H43W significantly inhibited mitochondrial fragmentation induced by these stressors evidenced by more fused and interconnected mitochondrial networks (p<0.05) compared to MARCH5 expressing cells. In addition, MARCH5H43W expression significantly (p<0.05) prevented mitochondrial membrane potential depolarization, which is considered a decisive event of mitochondrial failure. At last, intracellular ROS levels were evaluated by using CellROX-DeepRed staining and quantitative confocal imaging. Consistently, MARCH5H43W expression was able to lower the ROS production significantly following neurotoxic treatments. To further investigate the role of ubiquitin-dependent proteasome-mediated mitochondrial QC in neuronal cells, the impact of p97 inactivation in SH-SY5Y cell treated with neurodegenerative stressors such as rotenone, 6-hydroxydopamine, and amyloid-beta peptide (25-35) was analyzed. Compared to p97 expressing cells, p97QQ expressing cells displayed increased mitochondrial fragmentation, with additive effects observed during neurotoxic stress (p<0.01). Mitochondrial membrane potential level, as detected by TMRE (tetramethylrhodamine, ethyl ester) and evaluated by flow cytometry was significantly decreased following p97QQ expression. Again additive effects were found under neurotoxic stress conditions (p<0.05). ROS production was evaluated using the mitochondrial specific dye MitoSOX. A significant increase of mitochondrial ROS production was found in p97QQ expressing cells with or without treatments as compared to p97. In parallel, OxyBlot further confirmed increased levels of oxidized mitochondrial proteins in p97QQ expressing cells (p<0.05). Targeted mitochondrial autophagy or mitophagy occurs when mitochondrial damage cannot be dealt with by the molecular repair capacity degrading the entire, damaged organelle for recycling. Mitochondrial translocation of the ubiquitin ligase Parkin is an important step in this process. Thus, Parkin mitochondrial translocation assays were used to evaluate potential induction of mitophagic degradation under mild stress conditions and to further support the involvement of the proteasomal degradation system as part of mitochondrial QC. 1.5 Conclusions: Healthy and steady mitochondrial dynamics and QC machinery maintain mitochondrial function and subsequently support neuronal cell survival during endo- and/or exogenous neurotoxic stresses. Thus, understanding mitochondrial QC and its failing will yield important insight into the mechanisms of neurodegeneration.